Methods of treating cancer using immunostimulatory oligonucleotides

• US 20040087538A1
• Filed: 11/21/2003
• Published: 05/06/2004
• Est. Priority Date: 07/15/1994
• Status: Active Grant

• Current Assignee: United States As Represented By The Secretary Department of Health and Human Services, University of Iowa Research Foundation
• Original Assignee: University of Iowa Research Foundation
• Inventors: Krieg, Arthur M., Weiner, George
• Granted Patent: US 8,008,266 B2
• Time in Patent Office: Days
• US Class Current: 514/44
• CPC Class Codes: A61K 2039/55561 CpG containing adjuvants; Oligonucleotide containing adjuvants
• CPC Class Codes: A61K 31/00 Medicinal preparations containing organic active ingredients
• CPC Class Codes: A61K 31/4706 4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
• CPC Class Codes: A61K 31/7048 having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin , digitoxin or digoxin
• CPC Class Codes: A61K 31/711 Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
• CPC Class Codes: A61K 31/7125 Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 39/39 characterised by the immunostimulating additives, e.g. chemical adjuvants
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/02 Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 33/00 Antiparasitic agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 37/08 : Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 21/00 : Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/117 : Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
• CPC Class Codes: C12N 2310/17 : Immunomodulatory nucleic acids
• CPC Class Codes: C12N 2310/315 : Phosphorothioates
• CPC Class Codes: C12Q 1/68 : involving nucleic acids
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

Nucleic acid sequences containing unmethylated CpG dinucleotides that modulate an immune response including stimulating a Th1 pattern of immune activation, cytokine production, NK lytic activity, and B cell proliferation are disclosed. The sequences are also useful as a synthetic adjuvant.

Claims

1. An isolated nucleic acid sequence containing at least one unmethylated CpG dinucleotide and having a formula:

5′

N₁X₁CGX₂N₂3′

wherein at least one nucleotide separates consecutive CpGs;

X₁ is adenine, guanine, or thymine;

X₂ is cytosine or thymine;

N is any nucleotide and N₁+N₂ is from about 0-26 bases with the proviso that N₁+N₂ does not contain a CCGG quadmer or more than one CCG or CGG trimer; and

the nucleic acid sequence is from about 8-30 bases in length.

2. The nucleic acid sequence of claim 1, wherein X₁ is thymine

3. The nucleic acid sequence of claim 1, wherein X₂ is thymine.

4. The nucleic acid sequence of claim 1, which is GTCG (T/C) T or TGACGTT.

5. The nucleic acid sequence of claim 1, wherein the sequence is TGTCG (T/C) T.

6. The nucleic acid sequence of claim 1, which is TCCATGTCGTTCCTGTCGTT.

7. The nucleic acid sequence of claim 1, which is TCCTGACGTTCCTGACGTT.

8. The nucleic acid sequence of claim 1, which is TCGTCGTTTTGTCGTTTTGTCGTT.

12. A nucleic acid sequence of any of claims 1 or 9, wherein at least one nucleotide has a phosphate backbone modification.

13. The nucleic acid sequence of claim 12, wherein the phosphate backbone modification is a phosphorothioate or phosphorodithioate modification.

14. The nucleic acid sequence of claim 13, wherein the phosphate backbone modification occurs at the 5′

end of the nucleic acid.

15. The nucleic acid sequence of claim 14, wherein the modification occurs at the first two internucleotide linkages of the 5′

end of the nucleic acid.

16. The nucleic acid sequence of claim 13, wherein the phosphate backbone modification occurs at the 3′

end of the nucleic acid.

17. The nucleic acid sequence of claim 16, wherein the modification occurs at the last five internucleotide linkages of the 3′

end of the nucleic acid.

18. A method of stimulating immune activation in a subject, wherein the stimulation is predominantly a Th1 pattern of immune activation, comprising administering to the subject a nucleic acid sequence having the formula of claim 1 or claim 9.

19. The method of claim 18, wherein the subject is human.

20. A method of stimulating cytokine production in a subject comprising administering to the subject a nucleic acid sequence having the formula of claim 1 or claim 9.

21. The method of claim 20, wherein the cytokine is selected from the group consisting of:

IL-6, IL-12, IFN-γ

, TNF-α and

GM-CSF.

22. The method of claim 20, wherein the subject is human.

23. The method of claim 20, where the nucleic acid sequence is selected from the group consisting of:

24. A method of stimulating NK lytic activity in a subject comprising administering to the subject a nucleic acid sequence having the formula of claim 1 or claim 9.

25. The method of claim 24, where the subject is human.

26. The method of claim 24, where the nucleic acid sequence is selected from the group consisting of:

27. A method of stimulating B cell proliferation in a subject, comprising administering to the subject a nucleic acid sequence having the formula of claim 1 or claim 9.

28. The method of claim 27, where the subject is human.

29. The method of claim 27, where the nucleic acid sequence is selected from the group consisting of:

30. A method of stimulating immune activation in a subject comprising administering to a subject an nucleic acid sequence having the formula of claim 1, wherein the nucleic acid sequence acts as an adjuvant.

31. The method of claim 30, where the subject is a mammal.

32. The method of claim 30, where the nucleic acid sequence is selected from the group consisting of:

33. A method for treating a subject having an asthmatic disorder by administering to the subject an nucleic acid sequence in a pharmaceutically acceptable carrier having the formula of claim 1.

34. The method of claim 33, where the subject is human.

35. The method of claim 33, where the nucleic acid sequence is TCCATGACGTTCCTGACGTT.

9. An isolated nucleic acid sequence containing at least one unmethylated CpG dinucleotide and having the formula:

5′

NX₁X₂CGX₃X₄N3′

wherein at least one nucleotide separates consecutive CpGs;

X₁X₂ is selected from the group consisting of GpT, GpG, GpA, ApT and ApA;

X₃X₄ is selected from the group consisting of TpT or CpT;

N is any nucleotide and N₁N₂ is from about 0-26 bases with the proviso that N₁ and N₂ does not contain a CCGG quadmer or more than one CCG or CGG trimer; and

the nucleic acid sequence is from about 8-30 bases in length.

10. The nucleic acid sequence of claim 9, wherein the nucleotide that separates at least two consecutive CpGs is thymine.

11. The nucleic acid sequence of claim 9, wherein X₃ and X₄ are thymine.

36. A method for treating a subject having an autoimmune or other CpG associated disorder by inhibiting CpG-mediated leukocyte activation comprising administering to the subject an inhibitor of endosomal acidification in a pharmaceutically acceptable carrier.

37. The method of claim 36, where the subject is human.

38. The method of claim 36, where the inhibitor is selected from the group consisting of:

bafilomycin A, chloroquine, and monensin.

39. The method of claim 38, where the inhibitor is administered at a dosage of the less than about 10 μ

M.

40. The method of claim 36, wherein the disorder is selected from the group consisting of systemic lupus erythematosus, sepsis, inflammatory bowel disease, psoriasis, gingivitis, arthritis, Crohn'"'"'s disease, Grave'"'"'s disease and asthma.

41. The method of claim 40, where the disorder is systemic lupus erythematosus.